The Effects of Angiotensin II and Captopril on Expression of Morphine Withdrawal Signs in Rat

The mechanisms of drug dependence and rewarding properties of opiates are not exactly known and several neurotransmitters seem to be involved. It is possible that the rennin-angiotensin system could interact with the opioid system, since it has been shown that angiotensin II(Ang) and ACE inhibitors have analgesic, anticonvulsant and antidepression effects and in some cases they could antagonize the effect of morphine. In the present study, the effect of Ang II and captopril on withdrawal signs was evaluated. Male wistar rats were anesthetized and i.c.v cannula implanted and allowed to recover from surgery. Morphine was injected (i.p, 3 times a day) for 4 days to induce morphine dependence. The animals were divided into 3 groups and received saline, captopril, Ang II, and i.c.v, before naloxone injection. Naloxone precipitated morphine withdrawal signs, compared to the morphine dependent rats in saline, captopril and Ang II groups. Results showed that in the captopril group, some of the withdrawal signs were significantly lower than the saline group (p<0.05 and p<0.01). In the Ang II group, some of the withdrawal signs were greater than the saline group (p<0.01 and p<0.001). Considering the fact that captopril can reduce endogenous opioid degradation, it could probably reduce the morphine withdrawal signs in this way. On the other hand, captopril and Ang II can interact with dopamine, serotonin, substance p, acetylcholine or nitric oxide in different brain regions and alter morphine withdrawal signs.

aDepartment of Physiology, School of Medicine, Mashhad
University of Medical Sciences, Mashhad, Iran. bDepartment of Physiology, School
of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Abstract

The mechanisms of drug dependence and rewarding properties of opiates are not
exactly known and several neurotransmitters seem to be involved. It is possible
that the rennin-angiotensin system could interact with the opioid system, since
it has been shown that angiotensin II(Ang) and ACE inhibitors have analgesic,
anticonvulsant and antidepression effects and in some cases they could
antagonize the effect of morphine. In the present study, the effect of Ang II
and captopril on withdrawal signs was evaluated.

Male wistar rats were anesthetized and i.c.v cannula implanted and allowed to
recover from surgery. Morphine was injected (i.p, 3 times a day) for 4 days to
induce morphine dependence. The animals were divided into 3 groups and received
saline, captopril, Ang II, and i.c.v, before naloxone injection. Naloxone
precipitated morphine withdrawal signs, compared to the morphine dependent rats
in saline, captopril and Ang II groups.

Results showed that in the captopril group, some of the withdrawal signs were
significantly lower than the saline group (p<0.05 and p<0.01). In the Ang II
group, some of the withdrawal signs were greater than the saline group (p<0.01
and p<0.001).

Considering the fact that captopril can reduce endogenous opioid degradation, it
could probably reduce the morphine withdrawal signs in this way. On the other
hand, captopril and Ang II can interact with dopamine, serotonin, substance p,
acetylcholine or nitric oxide in different brain regions and alter morphine
withdrawal signs.

Keywords: Captopril; Angiotensin II; Morphine withdrawal; Rat.

Introduction

The dopaminergic mesolimbic system that consist of ventral tegmental area (VTA),
nucleus accumbenc and medial prefrontal cortex, is considered to be crucial in
the rewarding actions of opiates and involved in drug dependence (1, 2).
Previous studies have found that the effect of angiotensin II (Ang II) on
learning and memory was abolished by a dopaminergic antagonist (pimozid)(3). In
addition, disruption of the dopaminergic endings in discrete structure of the
dopaminergic mesolimbic system confirmed the involvement of dopaminergic system
on angiotensins facilitation of learning and memory (3, 4). These data indicate
that most cognition-improving effects of Ang II could be through the activation
of dopaminergic mesolimbic system (3).

The renin-angiotensin system (RAS) was initially described as a circulating
humoral system influencing blood pressure, as well as the fluid and electrolyte
homeostasis (5). But now it is well known that an independent RAS exists in the
brain (6). Brain has its own intrinsic RAS, with all its components, and is
capable of synthesizing angiotensin peptides and components of this system (5,
6). It has also been proposed that Ang II plays a neurotransmitters role in the
central nervous system (CNS) (7). Ang II is involved in the regulation of other
neurotransmitters such as GABA (8), noradrenaline, and 5-hydroxytryptamine
(5-HT), as well as the inhibition of acetylcholine release (6). The angiotensin
AT1 antagonist, losartan, was found to abolish the Ang II induced improvement in
object recognition, thus the cognition?improvement effects of Ang II may be
transmitted by AT1 (9). However, subsequent contradictory findings showed that
losartan was also able to facilitate spatial and short-term memory, and could
also reverse the scopolamine?induced cognitive deficits (10).

Angiotensin converting enzyme (ACE) inhibitors, such as captopril, enhance
learning in rats and support the hypothesis that Ang II suppression may have
cognitive enhancing effects (11). Experiments showed that Ang II inhibits
acetylcholine release (12). Therefore, administration of ACE inhibitors could
enhance acetylcholine release, and this effect may be responsible for the
cognitive improvement (12).

The research for endogenous substances with anti-opioid activity has provided
several evidences for morphine tolerance and morphine addiction. Among several
of anti-opioid substances, cholecystokinin octapeptide (CCK-8) and Ang II are
probably most attractive in CNS. Both of the small peptides have an abundant and
widespread distribution in CNS. Ang II showed an anti-opioid activity as well as
reversed morphine-induced analgesia in rats (13).

For example Ang II, injected icv, exerted a dose-dependent anti-nociceptive
effect in the acetic acid-induced abdominal constriction test in mice (14) or
Ang II, administered intra-thecally, induced short lasting antinocicptive effect
in the rat (15). It is assumed that these effects are realized through an opioid
mechanism and activation of AT1 receptor (16). These data suggest the
participation of Ang II in transmission of nocicepive information and its
interaction with opioid receptors (16). I.c.v administration of Ang II produced
anti-nociceptive effects that could be blocked by pretreatment with naloxone
(16).

Several evidences have shown that ACE inhibitors reduce the degradation of
opioids and increase their level in the brain (17). In addition, ACE inhibitors
have been reported to increase general health, vitality and work performance. A
possible mechanism could be the release of beta-endorphins (18). It has been
suggested that ACE inhibitors can alter the dopamine level in the brain (19) and
beneficial effects of these drugs on Parkinson disease has been shown (19). The
effect of ACE inhibitors on learning and memory (20, 21) have been investigated
and shown that these effects are blocked by naloxone (22, 23). ACE inhibitors,
commonly used to treat hypertention, are also used in the treatment of
cocaine-abusing populations, based on their potential to reduce cocaeine use by
modulating the levels of dopamine and corticotrophin releasing factor in the
brain (24).

Due to this evidence, further studies are needed to be carried out to elucidate
the role of RAS in opiate reward and dependence. Therefore, in the present study
we evaluated the effect of Ang II (main product of RAS) and captopril (ACE
inhibitor) on morphine withdrawal signs in rat.

Experimental

Animals and drugs

Male wistar rats weighing 200-250 g (Razi institute, Tehran, Iran) were used in
the present study. Animals were housed four to five per cage, with free access
to food and water ad libitum, and maintained at 22.0?2.0 ?C on a 12-light/dark
cycle (light period 07:00 and 19:00 h). All the animals were allowed with the
adapt to laboratory conditions for at last 1 week. The Isfahan University
committee on animal research approved the experiments.

The drugs used were morphine (TEMAD Ltd., Teheran, Iran), Ang II (Sigma Co.,St
Louis,USA), and captopril and naloxone (Daroo-Pakhsh Pharma, Iran). All the
drugs were dissolved in saline solution.

Intra-cerebroventricular cannula implantation

Animals were anaesthetized with ketamine (150 mg/kg, i.p) and rampon (0.1 mg/kg,
i.p) (25), then placed in a stereotaxic instrument (Stolting Instruments, USA).
Stainless steel, 23-gauge-guide, cannula was implanted 1 mm above the right
lateral cerebral ventricle. Sterotaxic coordinates were according to rat brain
atlas of Paxinos and Watson (26) (0.9 mm posterior to the bregma, lateral+1.6 mm
lateral to the sugittal suture and 3 mm from the top of skull). Cannula was
fixed with dental acrylic cement, anchored by two screws placed in the skull. A
stylet (26-gauge stainless steel) was placed into the guide cannula to allow the
guide cannula to maintain patency. After surgery, rats were given 300,000 units
of procaine penicillin G (i.p) to prevent infection. Animals were allowed 7 days
to recover from surgery (27).

Intra-cerebroventricular injection procedure

For drug injection, the rats were gently restrained by hand. The stylet was
removed from the guide cannula and a 27-gauge injection needle (1 mm beyond the
tip of the implanted guide cannula) was inserted. The injection needle was
attached to a 10 μl Hamilton syringe by a polyethylene tube. The injection
solutions were administered as a total volume of 5 μl. The injection needle was
retained in the guide cannula for an additional 60 s after injection, to
facilitate diffusion of the drugs (28).

Procedures

To induce morphine dependence, morphine was administered i.p. (3 injections each
day at 150 min intervals) for 4 days, in doses of 9, 16 and 25 mg/kg (1st day);
25, 25 and 50 mg/kg (2nd day), 50, 50 and 50 mg/kg (3rd day) and 50, 50 and 100
mg/kg (4th day). 180 min after the last morphine injection, the animals received
naloxone (3 mg/kg i.p) and the signs of morphine withdrawal were measured (29).
Ang II (1 nmol per rat) and captopril (300 μ/rat) were administered (i.c.v) 5
and 30 min before the naloxone injection to captopril and Ang II groups,
respectively. Then the animals were placed in plastic cylinders (50?18 cm) and
the following signs were observed and evaluated for 30 min: the number of
jumping, wet dog shakes, writhing, teeth chattering, grooming, genital grooming,
standing and the percentage of weight loss before and 30 min after naloxone
injection.

Experimental design

To evaluate the effects of captopril and Ang II on morphine withdrawal signs, 27
male rats (250-300 g) were used. The animals were divided into 3 groups:
1-saline group, which received morphine during 4 days and then received saline
(5μl i.c.v) at the end of experiment (4th day) 30 min before the naloxone
injection to precipitate withdrawal symptoms. 2-Captopril group, which received
morphine for 4 days, and then received captopril (300 μg i.c.v 30 min before
naloxone. 3- Ang II group, which received morphine for 4 days, and then received
Ang II (1 nmol in 5 μl of saline i.c.v) 5 min before naloxone. At the end of
experiment, all animals (on the 4th day) received naloxone (3 mg/kg, i.p) and
withdrawal signs were recorded for 30 min.

Data analysis

The overall treatment effects of the experiments were examined using a one-way
analysis of variance (ANOVA) and post-hoc comparisons. The criterion for
statistical significance was p<0.05.

Histology

Immediately after the tests, all the rats were given 2 μl of methylene blue in a
lateral ventricle, anesthetized with a high dose of anesthetic and perfused with
100 ml of saline followed by 100 ml of formalin (10%) transcardially. The brains
were removed and placed in formalin (10%). After 3 days, the brains were sliced
into 60-μm-thin slices. Data from rats with incorrect placement were excluded
from the analysis (30).

Results and Discussion

As shown in Figure 1, in the captopril group the number of standing, grooming
(Figure 1A) and writhing (Figure 1B), were significantly (p<0.05 and p<0.01)
lower than that of the saline group (p<0.01). Number of jumping was lower than
that of the saline group, but the difference was not significant (Figure 1B).
This shows that i.c.v injection of captopril could reduce morphine withdrawal
signs in rats. The number of teeth chattering and wet dog shakes in the Ang II
group was significantly greater than the morphine group (Figure 1A, p<0.01 and
p<0.001). There was no significant difference in the percentage of weight loss
in the 3 groups (Figure 1A).

In the present study, captopril injection (i.c.v 300 μg) 30 min before the
testing period, caused a decrease in morphine withdrawal signs (Figure 1). These
findings are in agreement with the previous studies, which have shown that i.c.v
injection of captopril in doses of 100, 300, 500 and 1000 μg could induce a dose
dependent anti-nociceptive effect in rats, this effect was completely blocked by
naloxone (10 mg/kg, i.p) (31). In the same study, it has been indicated that
i.c.v administration of 300 μg of captopril also potentiated the
anti-nociceptive effect of morphine in intact animals (31). Others have show
that the anti-nociceptive effect of repeated doses of captopril and losartan
were reversed by naloxone (32). It has been suggested that potentiation of
morphine-induced anti-nociception by captopril is unlikely to be exerted through
an effect on adrenal function and is most likely due to an increased brain
endogenous opioid system (31). In our study, a decrease in withdrawal signs
after injection of captopril may be due to the activation of endogenous opioid
system in the brain. In several studies it has been shown that ACE inhibitors
can activate this system (31). It is possible that this reduced withdrawal signs
of morphine after captopril injection, could be influenced by the concentration
of enkephalin in the brain. Other researchers have suggested that morphine could
increase in a concentration dependent manner, the degradation of leu- enkephalin
in bovine aortic endothelial cells. The enhanced leu- enkephalin degradation was
due to an increase in the activity of ACE (33). On the other hand, it has been
reported that endogenous opioid can reduce the withdrawal signs (34).

ACE can degrade substance P (17). We suggest that ACE inhibitors such as
captopril may reduce degradation of substance P, thus increasing it in the
brain. This could be another reason for a decrease in withdrawal signs. The
others have confirmed the hypothesis that substance P could abolish morphine
addiction in rats (35).

The effect of cholinergic system in morphine dependence has been investigated
and proposed that an increase in cholinergic system activity in the brain can
reduce morphine withdrawal signs (36). Decrease in morphine withdrawal by
captopril may be related to an increase in acetylcholine in the brain.

Preliminary experiments with captopril, 0.3 mg/kg s.c, enhanced some of the
naloxone-precipitated withdrawal signs such as rhinorrhea; lacrimation and
salivation, but other withdrawal sign were not altered by either captopril
treatment (37). But in our labratoy, captopril decreased some of the withdrawal
signs such as standing, grooming, writhing and increased wet dog shake, which
could be due to a difference in the method of administration.

The ACE activity in the rat brain was found to be decreased after implantation
of morphine pellets, that was abolished by naloxone (38). This shows the
existence of a permissive interaction between morphine and the renin-angiotensin
system.

It has been reported that Ang II and its fragments have cognition-improving
effects and facilitation of learning and memory process through an activation of
the central dopaminergic and glutamergic systems in the mesolimbic and
hippocampus (3). On the other hand it has been shown that the glutamergic and
dopaminergic system in these regions have important roles in the opiate
dependence (39, 40). Injection of Ang II (1 nmol) in morphine dependent rat
could increase some withdrawal signs such as teeth chattering and yawing (Figure
1A).

Administration of Ang II [(i.c.v) (14) and intrathecally (16)] exerted
anti-nociceptive effects that could be blocked by naloxone (16). It seems that
these effects are realized through an endogenous opioid mechanism (16) and
sometimes these effects were short lasting (16). Ang II also showed an
anti-opioid activity, which is reversed by morphine-induced analgesia in rats
(13).

Different mechanisms of the neuromodulatory action of Ang II on GABA release
have been discussed (41). It is possible that in our investigation Ang II could
interact with GABAergic system in the brain and increase some withdrawal signs
in this way.

It has been shown that CRH can increase morphine withdrawal signs (42). on the
other hand, it has been demonstrated that Ang II could increase CRH in the brain
and captopril decreases it (10, 43). Increased morphine withdrawal signs by Ang
II and it?s decrease by captopril in the present study, may be due to an
increase and decrease in CRH release, respectively.

We suppose that an important site for the effect of Ang II in our study may be
locus coeruleus (LC) nucleus, which contains considerable receptors for Ang II
(44, 45) and has the most important role in opioid withdrawal (46).

Another mechanism could be the alteration in dopaminergic system activity. There
are several evidences that RAS can alter dopaminergic activity in the brain (3,
7, 47, 48). The importance of this system in the rewarding property of morphine
and drug dependence has been established (30, 49), but it is controversial and
needed to be further investigated.

Finally, we propose that renin-angiotensin system could be involved in morphine
dependence, but the mechanism need to be further investigated.

Acknowledgment

Thanks are due to the Vice Presidency of Research of Mashhad University of
Medical Sciences, for financial assistance.